All animal experiments were carried out in accordance with the guidelines laid down by the animal welfare committees and the ethics committees of Niigata University. Mice deficient in γ-2 or γ-7 were produced by homologous recombination using the C57BL/6N ES cell line RENKA (Mishina & Sakimura, 2007). We isolated γ-2 (Cacng2) and γ-7 (Cacng7) genes by screening the genomic DNA library derived from the C57BL/6 mouse, and each gene fragment was yielded by PCR and sequenced. A γ-2 targeting vector contained exons 3 and 4 of Cacng2 gene with the 6.8-kb upstream and 4.8-kb downstream homologous genomic DNA fragments and the diphtheria toxin
gene for negative selection (Fig. 1A). A DNA fragment, which carried a 34-bp loxP sequence and pgk-1 promoter-driven Akt inhibitor neomycin phosphotransferase gene (pgk-neo) flanked by two Flp recognition target
(frt) sites, was inserted into the site 158 bp upstream of exon 3. The other loxP site was introduced into the site 159 bp downstream of exon 3 in order to Belnacasan supplier eliminate the exon 3 containing the two putative transmembrane domains after Cre-mediated recombination. The γ-7 targeting construct contained exons 1–3 of the Cacng7 gene with the 6.6 kb upstream, 4.5 kb downstream homologous genomic DNA (Fig. 1C). The loxP sequence and pgk-neo flanked by two frt sites was inserted into the site 340 bp upstream of exon 2. The other loxP site was introduced into the site 54 bp downstream of exon 3 in order to eliminate exons 2 and 3 after Cre-mediated recombination. Homologous recombinants were identified by Southern blot analysis under the following conditions: Kpn I-digested DNA hybridized with γ-2-5′ probe, 8.7 kb for wild-type (WT) and 8.2 kb for targeted allele; EcoR V-digested DNA hybridized with γ-2-3′ probe, 12.2 kb for WT and 10.2 kb for targeted allele; EcoR I-digested DNA hybridized with neo probe, 7.2 kb for γ-2-targeted allele; Spe I-digested DNA hybridized with γ-7-5′ probe, 20.3 kb for WT and 16 kb for targeted allele; EcoR I-digested DNA hybridized with γ-7-3′ probe, 8.2 kb for WT and 9.3 kb
for targeted allele; Hinc II-digested DNA hybridized with neo probe, 11 kb for γ-7-targeted allele. ES cell clones with correct recombination were used to yield chimeric mice as described see more previously (Fukaya et al., 2006). Chimeric mice were mated with C57BL/6 mice, and offspring were further crossed with TLCN-Cre mice (Nakamura et al., 2001; Fuse et al., 2004) to yield heterozygous KO mice (Fig. 1B and D). Homozygous γ-2- and γ-7-KO mice were obtained by crossing heterozygous pairs. The first offspring was genotyped by Southern blotting under the following conditions: EcoR I-digested DNA hybridized with γ-2-inner probe, 7.2 kb for WT and 6.7 kb for KO allele; EcoR I-digested DNA hybridized with γ-7-3′ probe, 8.2 kb for WT and 7.2 kb for KO allele.